Journal: BMC Neuroscience

Article Title: Chronic exposure to high fat diet triggers myelin disruption and interleukin-33 upregulation in hypothalamus

doi: 10.1186/s12868-019-0516-6

Figure Lengend Snippet: List of antibodies in the study for immunofluorescence or western blot analysis

Article Snippet: Polyclonal goat anti-IL-33 , R and D Systems Cat#AF3626, RRID:AB_884269 , Escherichia coli -derived recombinant mouse IL-33 , 1:200 (IF).

Techniques: Immunofluorescence, Western Blot, Purification, Affinity Chromatography, Recombinant, Derivative Assay

Journal: bioRxiv

Article Title: Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA

doi: 10.1101/2021.07.08.451582

Figure Lengend Snippet: RITA induces replication stress in SETD2-deficient cancer cells. (A) Confocal microscopy of phospho-RPA Ser33 (red) and 53BP1 (green) foci in U2OS cells after RITA treatment. Representative images from 3 independent experiments are shown. (B) Quantification of the average number of foci per nucleus from (A) . At least 70 nuclei per biological replicate (n = 3) were counted for statistical analysis. Data shown as mean ± SD. P-values were calculated using the two-tailed Student’s t-test. *p < 0.05, **p < 0.01. RITA induces replication stress in SETD2-deficient cancer cells. (C) DNA fibre assay of parental and SETD2-CRISPR U2OS. Cells were treated as indicated. Representative images from 2 independent experiments are shown. (D) Quantification of replication fork velocity from (C) . At least 200 fibres per biological replicate (n = 3) were measured for statistical analysis. Data shown as median + interquartile range. P-values were calculated using the two-tailed Student’s t-test. N.S. = non-significant, ****p < 0.000001.

Article Snippet: The primary antibodies used for immunofluorescence are: phospho-RPA (Ser33) (Bethyl Laboratories), 53BP1 (GeneTex), and γH2AX (Novus).

Techniques: Confocal Microscopy, Two Tailed Test, CRISPR

Journal: bioRxiv

Article Title: Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA

doi: 10.1101/2021.07.08.451582

Figure Lengend Snippet: Loss or inhibition of the phenol sulphotransferase SULT1A1 abolishes RITA-induced phenotypes. (A) Dose response viability curves for SETD2-CRISPR U2OS cells treated with RITA after the addition of non-targeting control (siNT) or SULT1A1 siRNA (siSULT1A1). Data are shown as mean ± SD. (B) Western blot of SULT1A1 in U2OS cells in the presence of non-targeting control (siNT) or SULT1A1 siRNA (siSULT). (C) Dose response viability curves for U2OS cells treated with RITA in the presence or absence of DCNP. Data are shown as mean ± SD. (D) Clonogenic survival assay for U2OS cells in the presence of the indicated compounds. Data are shown as mean ± SD. P-values were calculated using the two-tailed Student’s t-test. **p < 0.01, ****p < 0.0001. (E) Western blot of total p53, phospho-p53 (Ser15), and γH2AX in U2OS cells after the indicated treatments. (F) Western blot of procaspase 3 and cleaved caspase 3 in U2OS cells after the indicated treatments. (G) Cell cycle profiles of U2OS cells in the presence of the indicated compounds. Data shown as mean ± SD. (H) Confocal microscopy of phospho-RPA Ser33 (red) and 53BP1 (green) foci in U2OS cells after the indicated treatments. Representative images from 3 independent experiments are shown. (I) Quantification of the average number of foci per nucleus from (I) . At least 80 nuclei per biological replicate (n = 3) were counted for statistical analysis. Data shown as mean ± SD. P-values were calculated using the two-tailed Student’s t-test. *p < 0.05.

Article Snippet: The primary antibodies used for immunofluorescence are: phospho-RPA (Ser33) (Bethyl Laboratories), 53BP1 (GeneTex), and γH2AX (Novus).

Techniques: Inhibition, CRISPR, Control, Western Blot, Clonogenic Cell Survival Assay, Two Tailed Test, Confocal Microscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: Membrane tethering of CreER decreases uninduced cell labeling and cytotoxicity while maintaining recombination efficiency

doi: 10.1016/j.omtn.2022.01.022

Figure Lengend Snippet: mCreER does not leak into the nucleus Ad293 were transfected with pCMV-CreER-EGFP (A) or with pCMV-mCreER-EGFP (C). Tamoxifen (T) and/or rapamycin (R) were added 24 h after transfection. Microphotographs show the intracellular distribution of CreER-EGFP (A) and mCreER-EGFP (C) at 24 h and 48 h after induction. Scale = 20 μm. Quantitative analysis of EGFP percentage in the nucleus and leakiness of CreER-EGFP (B) and mCreER-EGFP (D) in Ad293 cells. In (B), white bars = uninduced, black bars = induced. EGFP leakiness was calculated as the ratio between uninduced vs. induced nucleus EGFP. Data are represented by mean ± standard error of the mean. n = 48. ∗∗∗ p < 0.001. In (D), Leakiness was calculated as the percentage of recombination rate of no induction (R-T-) or single induction (R + T-, R-T+) vs. that of dual induction (R + T+). Data are represented by mean ± standard error of the mean. n = 48. Bars denoted with a different letter on top are significantly different (p < 0.05). (E) Ad293 cells were transfected with pCMV-CreER , pCMV-mCreER , pCMV-mTEVp or pCMV-mCreER/pCMV-mTEVp . After induction for 0, 24, or 48 h, cells were stained with anti-Cre antibody (red) and Hoechst (blue), and analyzed by confocal microscopy. Scale = 50 μm. (F, G) Ad293 cells were transfected with pCMV-CreER (F), or with pCMV-mCreER/pCMV-mTEVp (G). Induction was carried out at 24 h after transfection. Western blots show the distribution of CreER in the membrane, cytoplasm or nucleus at 0, 24, or 48 h after induction. (H and I) Semi-quantitative analysis of the percentage of CreER in the membrane, cytoplasm or nucleus in (F) and (G), respectively. For densitometric analysis, cytoplasmic or membrane localized CreER at 0 h was set as 100%, while nuclear CreER at 48 h after induction was set as 100%. β-Actin, Na + /K + channel and PCNA were used for normalization and confirmation of the cytoplasm, plasma membrane and nucleus fractions, respectively. Data are represented by mean ± standard error of the mean. n = 6. In (D), (H) and (I), bars denoted with a different letter on top are significantly different (p < 0.05).

Article Snippet: Cells were incubated with the rabbit anti-H2A.X antibody (1:400, Cell Signaling Technology) or rabbit anti-Cre antibody (1:400, Cell Signaling Technology) for 2 h, and Cy3 goat anti-rabbit IgG (1:200, Beyotime, Shanghai, China) for 1 h, and finally with Hoechst 33,342 (10 μg/mL) (Beyotime, Shanghai, China) for 20 min. Three PBS washings were performed between each step.

Techniques: Transfection, Staining, Confocal Microscopy, Western Blot, Membrane, Clinical Proteomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: Membrane tethering of CreER decreases uninduced cell labeling and cytotoxicity while maintaining recombination efficiency

doi: 10.1016/j.omtn.2022.01.022

Figure Lengend Snippet: The dual inducible CreER system has low cytotoxicity Ad293 cells were transfected with pCMV-CreER , pCMV-mCreER , pCMV-mTEVp or pCMV-mCreER/pCMV-mTEVp . The control group was transfected with the vector backbone. Inducers were added at 24 h after transfection. Cells were analyzed at 0, 24, or 48 h after induction. (A) Cell viability was measured using the CCK-8 assay. (B–D) After staining with propidium iodine (PI), DNA content and cell cycle analysis was performed using flow cytometry. The proportion of cells in G1, S, and G2/M stages was determined using the Modfit software. Data are represented by mean ± standard error of the mean. n = 3. (E) γ-H2AX protein was detected by western blotting. (F) Semi-quantitative analysis of γ-H2AX protein density. Data are represented by mean ± standard error of the mean. n = 6. (G) Immunofluorescence staining of γ-H2AX (red) and Hoechst (blue) in transfected cells. (H) Semi-quantitative analysis of γ-H2AX signal. The relative intensity of γ-H2AX in cells expressing CreER at 48 h was set as 100 (black bar). Data are represented by mean ± standard error of the mean. n = 12. (I) Western blot analysis of P53 protein. (J) Semi-quantitative analysis of P53 protein density. The relative intensity of P53 (normalized with β-actin) in cells expressing CreER at 48 h was set as 100 (black bar). Data are represented by mean ± standard error of the mean. n = 3. (K) Quantitative real-time RT-PCR analysis of p53 transcript levels. β-Actin was used for normalization. Data are represented by mean ± standard error of the mean. n = 3. The significant difference to the control is denoted with ∗ p < 0.05, or ∗∗∗ p < 0.001; significant difference between CreER and mCreER/mTEVp is denoted with # p < 0.05.

Article Snippet: Cells were incubated with the rabbit anti-H2A.X antibody (1:400, Cell Signaling Technology) or rabbit anti-Cre antibody (1:400, Cell Signaling Technology) for 2 h, and Cy3 goat anti-rabbit IgG (1:200, Beyotime, Shanghai, China) for 1 h, and finally with Hoechst 33,342 (10 μg/mL) (Beyotime, Shanghai, China) for 20 min. Three PBS washings were performed between each step.

Techniques: Transfection, Control, Plasmid Preparation, CCK-8 Assay, Staining, Cell Cycle Assay, Flow Cytometry, Software, Western Blot, Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: Nutrients

Article Title: Caffeine and the Risk of Diabetic Retinopathy in Type 2 Diabetes Mellitus: Findings from Clinical and Experimental Studies

doi: 10.3390/nu15051169

Figure Lengend Snippet: ( A ) The effect of caffeine eye drops on glial activation. Upper panel: Comparison of GFAP immunoreactivity (green) in the retina among representative samples from a diabetic mouse treated with vehicle, a diabetic mouse treated with caffeine, and a non-diabetic mouse. Nuclei were labeled with Hoechst (blue). ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bars, 20 µm. Lower panel: Quantification of glial activation based on the extent of GFAP staining. N: 6 mice per group; ( B ) Effect of caffeine eye drops on vascular leakage. Upper panel: Confocal immunofluorescence images of vascular permeability assessed by Evans Blue dye leakage in retinal whole mounts. Spec3, fluorescent spectral signature 3. FI: fluorescence intensity. Scale bars, 20 μm. Lower panel: For quantification, the number of extravasations per field of 60× retina was counted. N: 4 mice per group.

Article Snippet: Nuclei were counterstained with the blue fluorescent DNA stain Hoechst 33,342 ( 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate ) (Thermo Fisher Scientific, OR, USA) after washing.

Techniques: Eye Drops, Activation Assay, Comparison, Labeling, Staining, Immunofluorescence, Permeability, Fluorescence

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RNAi screening identifies RABGAP1L as an IAV restriction factor (A) Schematic representation of recombinant IAV WSN/33 in which the coding region for the hemagglutinin (HA) glycoprotein has been replaced by Renilla luciferase (WSN/33- Renilla ). (B) RNAi-screening experimental workflow. (C) MRC-5-HA cells were transfected for 30 h with individual siRNAs targeting MX1 or IFITM3 or with a non-targeting (NT) control siRNA. Following stimulation with IFNα2 (1,000 U/mL or mock) for 16 h, cells were infected with WSN/33- Renilla (MOI 5 PFU/cell) in the presence of the live-cell substrate EnduRen. Luciferase activity was monitored up to 12 h post-infection (p.i.), and the area under the curve (AUC) was calculated as indicated. Mean values from 50 technical replicates across two independent biological experiments are plotted, with error bars representing SDs. (D) Hit criteria for RNAi screening. In a primary screen following the workflow in (B), 100 putative ISGs were silenced with four individual siRNAs each. Twenty-two genes met the threshold, and 20 were re-tested in a confirmation screen. Applying the same hit criteria, a total of 8 putative ISGs were confirmed in both screening rounds. (E) Heatmap showing Z scores of positive controls ( MX1 and IFITM3 ) and the top 8 hits from the two RNAi-screening rounds. Columns represent individual siRNAs targeting genes listed in rows. See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Luciferase, Transfection, Infection, Activity Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: IFN-mediated restriction of IAV by RABGAP1L (A) A549 cells were transfected with the indicated siRNAs for 32 or 60 h prior to lysis and assessment of cell viability using CellTiter-Glo. An NT siRNA and an siRNA targeting IRF9 were used as negative controls. siRPS is an siRNA targeting the essential gene RPS27A and thus acted as a positive control for cell toxicity. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (B and C) A549 cells were transfected with the indicated siRNAs 30 h prior to IFNα2 treatment (1,000 U/mL or mock). Sixteen hours post-IFN stimulation, cells were infected with WSN/33- Renilla (MOI 1 PFU/cell), and luciferase activity was monitored every 2 h for a total of 12 h. The NT siRNA and siRNA targeting IRF9 were used as controls. (C) The AUC was calculated from measured relative light units (RLUs) over time. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (D) In parallel to (B) and (C), cells were harvested for western blot analysis 16 h post-IFN stimulation. Proteins of interest were detected as indicated. RABGAP1L (RG1L) isoforms corresponding to detected bands are highlighted. (E) Schematic representation of RABGAP1L isoforms A, G, H, and I, showing the phosphotyrosine-binding (PTB) domain, the kinesin-like (kin) domain, and the Tre-2/Bub2/Cdc16 (TBC) domain. Isoform G further contains a domain of unknown function (DUF3084). (F) Immunofluorescence analysis of A549 cells stably expressing either empty vector (EV) or RABGAP1L isoforms A, G, H, and I. Cells were fixed and stained for RABGAP1L (red); nuclei were stained with DAPI (blue). Scale bar represents 25 μm. Representative confocal-microscopy images from at least two biologically independent experiments are shown. (G) Cells described in (F) were harvested for western-blot analysis. Proteins of interest were detected with the indicated antibodies. Images are representative of three biologically independent experiments. (H) Cells described in (F) and (G) were treated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected 48 h p.i. and titrated on Madin-Darby canine kidney (MDCK) cells to determine viral titers. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance in (C) and (H) was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001; ns, non-significant). See also and .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Transfection, Lysis, Positive Control, Infection, Luciferase, Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L overexpression restricts selected positive- and negative-sense RNA viruses (A) A549 cells stably expressing GFP or RABGAP1L (RG1L) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with different Renilla luciferase-encoding IAVs: H1N1 (WSN/33, MOI 1 PFU/cell), pdmH1N1 (Neth/09, MOI 5 PFU/cell), or H5N1 (Viet/04, MOI 0.5 PFU/cell). EnduRen live-cell substrate was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs up to 11 h p.i. (B) Huh-7 cells stably expressing GFP or RG1L were treated as described in (A) and infected with WSN/33- Renilla (MOI 1 PFU/cell) or HCoV-229E- Renilla (MOI 5 PFU/cell). EnduRen was supplemented, and the luciferase activity was measured every 2 h for a total of 11 h. RLUs were used to calculate the AUC. (C–E) A549 cells expressing EV or RG1L were stimulated with IFNα2 (10, 100 or 1,000 U/mL or mock) for 4 h prior to infection with VSV-GFP (MOI 1 PFU/cell) (C) or for 16 h prior to infection with SeV-GFP (MOI ∼1 PFU/cell) (D) and NDV-GFP (MOI 1 PFU/cell) (E). GFP intensity was measured every 2 h for up to 72 h. The AUC was calculated from total green integrated intensity. (F and H) Calu-3 (F) or Vero-CCL81 (H) cells stably expressing GFP or RG1L were treated with IFNα2 (10, 100, or 1,000 U/mL or mock) for 16 h, followed by infection with WSN/33- Renilla (MOI 1 PFU/cell). EnduRen was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs. (G and I) Calu-3 (G) or Vero-CCL81 (I) cells stably expressing GFP or RG1L were treated as described in (F) prior to infection with SARS-CoV-2 (MOI 0.1 PFU/cell). Supernatants were collected 24 h p.i., and viral titers were determined by plaque assay in Vero-E6 cells. (A–I) Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined comparing GFP-overexpressing with RG1L-overexpressing cells in equal treatment conditions in all panels using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.0002, ∗∗∗∗ p < 0.0001; ns, non-significant).

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Over Expression, Stable Transfection, Expressing, Infection, Luciferase, Activity Assay, Plaque Assay, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: The antiviral function of RABGAP1L relies on its catalytically active TBC domain and residues implicated in endosomal trafficking (A) Schematic representation of RG1L WT and the 421 mutant (RG1L 421) which lacks the C-terminal region downstream of the kin domain. (B) Immunofluorescence analysis of A549 cells stably expressing EV, RG1L WT, or RG1L 421. Cells were fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (C) A549 cells stably expressing GFP, RG1L WT, or RG1L 421 were stimulated with IFNα2 (1,000 U/mL or mock) for 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected after 48 h and titrated on MDCK cells. (D) Schematic representation of the TBC domain of RABGAP1L and the localization of mutants R584A (R mut ), Q621A (Q mut ), R584A-Q621A (RQ mut ), and KK784EE (KK mut ). KK mut has previously been shown to prevent interaction with the AnkB death domain (DD). (E) Western blot validation of RABGAP1L expression in A549 cells stably expressing RG1L WT or the indicated mutants. (F) Immunofluorescence analysis of cells described in (E) (here, EV was used as a control), fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (G) Cells described in (E) were infected with WSN/33- Renilla (MOI 1 PFU/cell) following treatment with IFNα2 (1,000 U/mL or mock) for 16 h. The AUC was calculated from RLU values taken up to 11 h p.i. For (B), (E), and (F), representative data from three biologically independent experiments are shown. For (C) and (G), mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001). See also Figure S3 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Mutagenesis, Immunofluorescence, Stable Transfection, Expressing, Staining, Infection, Western Blot, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: Proximity-labeling-based proteomics identifies the RABGAP1L host interactome (A) Schematic representation of TurboID-V5-tagged (T-V5) GFP (negative control) carrying a nuclear-export sequence (NES) or T-V5-tagged RABGAP1L (T-V5-RG1L). (B) Constructs described in (A) were stably expressed in A549 cells, and their expression was validated by immunofluorescence using an α-V5 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (C) Western blot analysis of cells described in (B) compared with A549 cells stably expressing untagged GFP or RABGAP1L (RG1L). Proteins of interest were detected with the indicated antibodies. (D) Cells described in (C) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33- Renilla (MOI 1 PFU/cell). The AUC was calculated from RLU values taken up to 11 h p.i. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (E) Workflow of the TurboID proximity-labeling approach. Cells described in (B) were treated with IFNα2 (1,000 U/mL or mock) for 16 h, followed by treatment with biotin (500 μM) for 15 min. Following streptavidin-based affinity purification, peptides were generated and subjected to mass-spectrometry analyses. (F) Interactors specific to RABGAP1L (as compared to GFP-NES) identified using the protocol described in (E). Hits are listed with their gene names and sorted according to previously described functions. Most hits were identified in non-IFNα2-treated samples. Hits marked with an asterisk ( ∗ ) were identified in the presence and absence of IFNα2, and hits marked in bold were only identified in IFNα2-treated samples. (G) A549 cells stably expressing constructs introduced in (A) or T-V5-tagged RABGAP1L KK mut and RQ mut were subjected to the proximity labeling approach outlined in (E). Following streptavidin-based affinity purification (samples termed “eluates”), total lysates and eluates were analyzed by western blot. Proteins were detected with the indicated antibodies. Data obtained in (B), (C), and (G) are representative of three biologically independent experiments. For (D), statistical significance was determined using one-way ANOVA following log transformation (ns, non-significant). See also and Figure S4 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Labeling, Negative Control, Sequencing, Construct, Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot, Infection, Affinity Purification, Generated, Mass Spectrometry, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L expression impacts host endosomal function and IAV uptake (A–C) A549 cells stably expressing RABGAP1L WT, the 421-truncation mutant, or EV were infected with WSN/33 (MOI 5 PFU/cell) for 1 h on ice. Three hours after incubation at 37°C, cells were fixed and stained with antibodies against RABGAP1L (red) and NP (green) (A). Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (B and C) Green mean fluorescent intensities (MFIs) of nuclear NP signals were quantified from fluorescent-microscopy images from (A) using ImageJ software. Individual cells are represented by single dots (B). Mean values of data from three biologically independent experiments in (B), normalized to EV, are shown in (C). (D) MDCK cells, expressing the constructs described in (A), were infected for 4 h at 37°C with WSN/33-pseudotyped β-lactamase-matrix protein (BlaM1) fusion protein VLPs prior to quantification of entry-positive cells via flow cytometry. Data represent means, with error bars showing SDs, from three biologically independent experiments. Individual data points are shown. (E) Experimental setup for immunofluorescence-based confocal microscopy to track early stages during IAV entry. Following infection with WSN/33 (MOI 25 PFU/cell or mock) for 1 h at 4°C, cells were fixed at the indicated timepoints. (F) A549 cells stably expressing RABGAP1L (RG1L) or EV were subjected to the experimental setup described in (E). The MFI of HA signals (green) at 0 min p.i. were quantified from confocal-microscopy images shown in Figure S6 A using ImageJ. Individual cells are represented by single dots. (G) Quantification of co-localizations between EEA1 and HA from confocal images shown in (H) and Figure S6 A using Imaris. Individual cells are represented by single dots. (H) Immunofluorescence analysis of RABGAP1L or EV-expressing A549 cells treated as described in (E). Cells were stained for early endosomes (EEA1, magenta), viral proteins (HA, green), and nuclei (DAPI, blue). Scale bar represents 25 μm. White arrows indicate co-localizations between EEA1 and HA. Representative images of at least nine analyzed cells per time point from at least two biologically independent experiments. (I) Quantification of co-localizations between EEA1 and HA from confocal images shown in Figure S6 B. Individual cells are represented by single dots. (J and K) Cells described in (A) were serum starved for 2 h prior to treatment with Dynasore (Dyn.; 100 μm) or DMSO for 1 h at 37°C. Cells were then incubated with Alexa-Fluor-488-conjugated transferrin (Tf-488) for 1 h at 4°C followed by a 10-min incubation at 37°C prior to fixation. (J) MFI quantification of Tf-488 signals from confocal-microscopy images shown in (K) using ImageJ software. Individual cells are represented by single dots. (K) Cells were stained with anti-transferrin receptor (TfR) antibody (magenta) and DAPI (blue) prior to analysis by confocal microscopy. Scale bar represents 25 μm. Representative images of at least 25 analyzed cells from two biologically independent experiments. Statistical significance was determined using unpaired nonparametric t test (B, F, G, and J), unpaired one-way ANOVA (C and D), or ordinary two-way ANOVA (I) ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, non-significant). See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Expressing, Stable Transfection, Mutagenesis, Infection, Incubation, Staining, Microscopy, Software, Construct, Flow Cytometry, Immunofluorescence, Confocal Microscopy

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Cell Viability Assay, Mutagenesis, Clone Assay, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 1. The list of differentially expressed genes in C33A/CA9 and C33A/Mock cells. Differentially expressed genes were selected on the basis of fold-change in CA9-overexpressed (C33A/CA9) cells versus vector- transfected control (C33A/Mock) cells. Gene expressions were confirmed by RT-PCR and quantitative RT-PCR. The relative quantity for each molecule was normalized with GAPDH, which was used as an internal control. Each bar represents the mean (plus standard error) of four independent experiments. *P<0.05.

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Plasmid Preparation, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 2. Reorganization of actin cytoskeleton and alteration of focal adhesion induced by CA9 overexpression. (A)C33A/CA9 and C33A/Mock cells were incubated on glass slides pre-coated with BD Matrigel Matrix with the cultured medium for 24 hours. Actin filaments (green) of cytoskeleton, vinculin (red) of focal adhesion complex and nuclear DNA (blue) were stained with phalloidin conjugated with Alexa 488, anti-vinculin antibody labeled with Alexa 594 and DAPI, respectively. (B)HeLa cells were cultured under normoxic (20% O2) and hypoxic (0.1% O2) conditions and were stained in the same manner as in A. (C)Cells grown as described above were harvested and CA9 expression was confirmed using M75 monoclonal antibody. Expression and phosphorylation of paxillin was examined by immunoblotting, using the indicated antibodies (anti-paxillin, anti-pPaxillinY118). -actin was used as a loading control.

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Over Expression, Incubation, Cell Culture, Staining, Labeling, Expressing, Phospho-proteomics, Western Blot, Control

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 4. Rho-GTPase activity and changes in cytoskeletal structure and focal adhesion with CA9 overexpression. (A)The amount of GTP-bound Rho-GTPase was compared between two cells by GST-RBD pull-down assay. Band intensity of activated RhoA/B/C-GTPases was quantified by image software, Multi Gauge, V3.0 (FUJIFILM). (B)Changes in actin cytoskeleton and focal adhesions subsequent to blocking the Rho downstream with ROCK inhibitor (Y27632) were observed by immunofluorescence analysis. C33A/Mock and C33A/CA9 cells were plated as in Fig. 2A and treated with a final 10M Y27632 after 3 hours. Actin filaments, vinculins and nuclear DNA were stained as green, red and blue signal, respectively. (C)Total lysates from two cell line cultures treated with or without 10M Y27632 as described in B were used for immunoblotting for expression and phosphorylation of paxillin. -actin was used as a loading control.

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Activity Assay, Over Expression, Pull Down Assay, Software, Blocking Assay, Immunofluorescence, Staining, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 3. Increased invasive and metastatic ability with CA9 overexpression. (A)C33A/CA9 and C33A/Mock cells were suspended as single cells and were cultured in Matrigel for two weeks. Images of the spheroid colonies were acquired using a light microscope (100 magnification). (B,C)Migratory and invasive ability of two cell lines were examined using Transwell chambers with or without Matrigel coating. The cells migrating through the filters were enumerated and the relative ratio of migrating C33A/CA9 cells to migrating C33A/Mock cells was determined. Each illustration shows the typical image and each bar represents the mean (plus standard error) of six independent experiments (*, P<0.05 compared with C33A/Mock cells).

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Over Expression, Cell Culture, Light Microscopy

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 5. Migratory and invasive potential improved with Rho-GTPase inactivation in C33A/CA9 cells. (A)C33A/Mock and C33A/CA9 cells were cultured in Matrigel for 8 days, as in Fig. 3A and were treated with final 10 M Y27632 at this time to start cells clumping. Y27632 inhibitor was treated two more times and the cells were observed using a light microscope (100 magnification). (B,C)Migrating and invading ability of the cells were examined as in Fig. 3B and 3C. Cells were seeded onto respective Transwell chambers and were treated with 10 M Y27632 3 hours after seeding. Each figure illustrates the typical image obtained and each bar represents the mean (plus standard error) of six independent experiments.

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Cell Culture, Light Microscopy

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 6. The role of Rho-GTPase signaling in the EMT process. (A)The expression and localization of E-cadherin and vimentin were examined by immunofluorescence analysis. Prepared under the same conditions as in Fig. 4B, the cells were stained for E-cadherin (red) and vimentin (green) with Alexa-594- and Alexa-488-labeled secondary antibodies, respectively. (B)Thirty micrograms of the membranous and cytoplasmic fraction of proteins obtained from the cells were loaded and the expression of E-cadherin was analyzed by western blotting. PMCA4 (plasma membrane calcium ATPase 4) and - tubulin were used as controls for membranous and cytoplasmic proteins, respectively. (C)Expression of epithelial markers (E-cadherin and -catenin) and mesenchymal markers (vimentin and N- cadherin) were examined by western blot in both C33A/Mock and C33A/CA9 cells, with or without Y27632 treatment. -actin was used as a loading control. Band intensity from western blot images was quantified by Multi Gauge V3.0 (FUJIFILM). Each bar represents the mean (plus standard error).

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Western Blot, Clinical Proteomics, Membrane, Control

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 7. Rho signaling is crucial to form the migratory phenotypes. The role of Rho signaling was validated using two types of siRNAs against RhoA at a concentration of 25 nM. A double-stranded non- specific siRNA was used as a negative control (CNT). (A)Block efficiency of si-Rho and quantitative changes of EMT markers were analyzed by western blot. -actin was used as a loading control. (B)An invasion assay using Transwell chambers with Matrigel coating was repeated six times. The cells migrating through the filters were enumerated and the relative ratio of invasive cells was normalized by the number of migrating C33A/Mock-CNT cells. Illustrations show the typical images and the bars represent the mean (plus standard error).

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Concentration Assay, Negative Control, Blocking Assay, Western Blot, Control, Invasion Assay

Journal: Journal of cell science

Article Title: Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

doi: 10.1242/jcs.072207

Figure Lengend Snippet: Fig. 8. The effect of CA9 on migration and invasion is validated by siRNAs against CA9. (A)Block efficiency of si-CA9 was validated by western blot. A double-stranded non-specific siRNA was used as a negative control (CNT). C33A/CA9 cells were transfected with 150 nM siRNA against CA9 for 24 hours and then were transferred onto Matrigel-coated dishes. After incubation for 24 hours, total cell lysates were analyzed for small GTPases such as Rho, RAC, CDC42 and paxillin using a pull-down assay and western blot analysis. Band intensity from western blot images was quantified by Multi Gauge V3.0. (B,C)At 24 hours post- transfection, the cells were prepared for migration and invasion assays as in Fig. 5B and C. (D)Cells were prepared as in Fig. 8A and were analyzed for EMT markers by western blot analysis. After silencing of CA9 expression, epithelial markers increased in expression, whereas mesenchymal markers decreased in C33A/CA9 cells. Each bar represents the mean (plus standard error).

Article Snippet: Cell culture and stable transfection of C33A cells The human cervical cancer cell line C33A (ATCC number HTB-31) was stably transfected with either full-length human CA9 cDNA cloned into the pcDNA3 vector or empty vector control (Shin et al., 2008).

Techniques: Migration, Blocking Assay, Western Blot, Negative Control, Transfection, Incubation, Pull Down Assay, Expressing

Journal: Nature Communications

Article Title: A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity

doi: 10.1038/ncomms9093

Figure Lengend Snippet: ( a ) Confocal microscopy revealed cells (labelled with arrows) with a heavily laden β-catenin (bcat) nuclear staining ( b ), in contrast to nearby PTEN-GFP-positive cells, where β-catenin staining could seldom be observed within the nucleus, arrowheads. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) ( c ). Merged is shown ( d ). Scale bar, 10 μm. Immunofluorescence experiments for the Hs944T cells were performed three times with similar results. ( e – h ) Human melanoma Lyse cells mutated for NRAS (Q61K), which produce PTEN, were transfected with siScr ( e , f ) and siPTEN ( g , h ). Cells were labelled for β-catenin ( e , g ) and counter stained with DAPI ( f , h ). Scale bar, 10 μm. Immunofluorescence experiments for the Lyse cells were performed two times with similar results. ( i , j ) Confocal microscopy showing the localization of β-catenin in Tyr::Cre/°;PTEN f/+ =PTEN f/+ ( i ) and Tyr::Cre/°;PTEN f/f =PTEN f/f ( j ) melanocytes. Note the increase of nuclear β-catenin staining in PTEN f/f cells. Scale bar, 10 μm. Immunofluorescence experiments for the murine PTEN f/+ and PTEN f/f cells were performed four times with similar results. ( k ) Immunoblot analysis of PTEN, AKT (total and phosphorylated form Ser473), GSK3β (total and phosphorylated form Ser9), β-catenin (total, phosphorylated form Thr41/Ser45 and Ser675) and β-actin proteins in Hs944T transfected with either expression vector encoding GFP (CMV::GFP) or PTEN (CMV::PTEN-GFP). Cells expressing either exogenous GFP or PTEN treated with LY294002 or LiCl for 1 h. It is noteworthy that a higher concentration of GSK3β antibody reveals a second upper band. Western blot analyses were performed two times for all antibodies with similar outputs. ( l ) Immunoblot blot analysis for GFP, PTEN, AKT (total and pSer473), β-catenin pSer675 and β-actin of Hs944T lysates co-transfected with either empty vector GFP or PTEN and WT or constitutively active p110 E545K mutant. Cells were starved for 2 h with 0.1% serum before lysis. Western blot analyses were performed two times for all antibodies with similar outputs. ( m ) Immunoblot blot analysis for PTEN, AKT (total and pSer473), β-catenin pSer675 and β-actin of Hs944T lysates co-transfected with a constitutive active form of p110 (p110 CAAX) or a kinase-dead form (p110 KD) in the presence of GFP or PTEN. Western blot analyses were performed three to six times, depending on the antibody with similar outputs.

Article Snippet: Membranes were probed with antibodies against ERK (Cell Signaling, 9102), pERK (Thr202/Thr204, Cell Signaling, 9106), CREB (Cell Signaling, 9192), pCREB (Cell Signaling, 9191S), PTEN (Cell Signaling, 9559), AKT (Cell Signaling, 2938), pAKT (Ser473, Cell Signaling 3787), GSK3-β (Santa-Cruz, sc-9166), pGSK3-β (Ser9, Cell Signaling, 9336), S6 (Cell Signaling, 2317), pS6 (Ser235/236) (Cell Signaling, 4857), β-catenin (Abcam, ab-6302), pβ-catenin (Ser675, Cell Signaling, 4176), pβ-catenin (Ser33-37/Thr41, Cell Signaling, 9561), pβ-catenin (Thr41/Ser45, Cell Signaling, 9565S), p16 (Santa-Cruz, sc-1661), CAV1 (Cell Signaling, 3238) and β-actin (Sigma, A5441).

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Transfection, Western Blot, Expressing, Plasmid Preparation, Concentration Assay, Mutagenesis, Lysis

Journal: Nature Communications

Article Title: A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity

doi: 10.1038/ncomms9093

Figure Lengend Snippet: ( a ) Interactome of PTEN and β-catenin (BCAT) as determined by the in silico Ingenuity Pathway Analysis (IPA). The Venn diagram reveals 63 common members. Interaction of CAV1 with β-catenin (BCAT; b ) and PTEN ( c ) in Rosi human melanoma cells. Extracts from ∼1.5 × 10 7 cells (corresponding to about 1.5 mg) were immunoprecipitated with control IgG, anti-BCAT, anti-CAV1 or anti-PTEN antibodies. Immune complexes were resolved by SDS–PAGE and blotted with antibodies to BCAT, CAV1 and PTEN. Total protein input corresponds to 2% of the total protein used for immunoprecipitation. One-tenth of the IP sample was loaded to detect BCAT following IP with BCAT antibodies (and similarly for CAV1 and PTEN). This allowed getting a reasonable intensity for the corresponding signals. For the other lanes, the entire IP samples were loaded. ( d ) GST pulldown using β-catenin–GST fusion Sepharose beads and whole-cell protein lysates (500 μg) from Hs944T cell transfection with GFP or PTEN. Pellet and supernatant fractions were immunoblotted for various antibodies. ( e ) CAV1-BCAT immunocomplex in GFP transiently transfected Hs944T cells. When transfected with PTEN, the proportion of β-catenin in the immunocomplex is dramatically reduced. Total protein input is shown. For b – e , experiments were performed three times. ( f – k ) Immunofluorescence of transiently transfected Hs944T human melanoma cell line with CMV::GFP and CMV::CAV1-RFP (CAV1) expression vector. β-Catenin staining ( f , i ), GFP ( g ), CAV1 ( j ) and merged ( h , k ). Scale bar, 25 μm. Immunofluorescence experiments for Hs944T cells were performed three times with similar results. ( l – t ) Immunofluorescence of mouse carcinoma submandibular gland (CSG) cells transfected with siRNA directed against either negative control (siScr), PTEN (siPTEN) and CAV1 (siCAV1) stained for BCAT ( l , o , r ), respectively. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) ( m , p , s ). Merge ( n , q , t ). Scale bar, 25 μm for all panels. Immunofluorescence experiments for the CSG cells were performed two times with similar results.

Article Snippet: Membranes were probed with antibodies against ERK (Cell Signaling, 9102), pERK (Thr202/Thr204, Cell Signaling, 9106), CREB (Cell Signaling, 9192), pCREB (Cell Signaling, 9191S), PTEN (Cell Signaling, 9559), AKT (Cell Signaling, 2938), pAKT (Ser473, Cell Signaling 3787), GSK3-β (Santa-Cruz, sc-9166), pGSK3-β (Ser9, Cell Signaling, 9336), S6 (Cell Signaling, 2317), pS6 (Ser235/236) (Cell Signaling, 4857), β-catenin (Abcam, ab-6302), pβ-catenin (Ser675, Cell Signaling, 4176), pβ-catenin (Ser33-37/Thr41, Cell Signaling, 9561), pβ-catenin (Thr41/Ser45, Cell Signaling, 9565S), p16 (Santa-Cruz, sc-1661), CAV1 (Cell Signaling, 3238) and β-actin (Sigma, A5441).

Techniques: In Silico, Immunoprecipitation, Control, SDS Page, Transfection, Immunofluorescence, Expressing, Plasmid Preparation, Staining, Negative Control

Journal: Nature Communications

Article Title: A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity

doi: 10.1038/ncomms9093

Figure Lengend Snippet: ( a ) TOP-FLASH activity in Hs944T cells in the presence of GFP, PTEN and/or CAV1 β-catenin (BCAT). ( b ) Similarly, TOP-FLASH activity was measured in the same cells post transfection of siRNA directed against negative control (siSCR), CAV1 (siCAV1), β-catenin (siBCAT). ( c ) Activity of p16INK4A::luciferase reporter was evaluated post transfection in Hs944T human melanoma cells with GFP, PTEN and/or CAV1 BCAT, and ( d ) with siSCR, siCAV1 and siBCAT. ( a – d ) All p16INK4A::luciferase and TOP-FLASH reporter assays were evaluated in the presence of an internal control ( Renilla luciferase). ( e , f ) p16 mRNA level as measured by quantitative reverse transcriptase–PCR (fold change), following overexpression of GFP, PTEN and/or CAV1 BCAT, or knockdown of CAV1 and BCAT, with appropriate controls. ( g ) Eighteen human melanoma tumours were stained for CAV1, PTEN and p16. PTEN and p16 were absent and CAV1 was present for patient 1. Opposite observation was performed for patient 2. Stromal and endothelial cells were used as positive control for PTEN and CAV1, respectively. Scale bar, 100 μm for all panels. Error bars represent s.d. * P -value <0.05, ** P -value <0.01 and *** P -value <0.001. Statistical significance was determined by Mann–Whitney test. Each experiment was performed in eight and three biological triplicates for a – d , e and f respectively.

Article Snippet: Membranes were probed with antibodies against ERK (Cell Signaling, 9102), pERK (Thr202/Thr204, Cell Signaling, 9106), CREB (Cell Signaling, 9192), pCREB (Cell Signaling, 9191S), PTEN (Cell Signaling, 9559), AKT (Cell Signaling, 2938), pAKT (Ser473, Cell Signaling 3787), GSK3-β (Santa-Cruz, sc-9166), pGSK3-β (Ser9, Cell Signaling, 9336), S6 (Cell Signaling, 2317), pS6 (Ser235/236) (Cell Signaling, 4857), β-catenin (Abcam, ab-6302), pβ-catenin (Ser675, Cell Signaling, 4176), pβ-catenin (Ser33-37/Thr41, Cell Signaling, 9561), pβ-catenin (Thr41/Ser45, Cell Signaling, 9565S), p16 (Santa-Cruz, sc-1661), CAV1 (Cell Signaling, 3238) and β-actin (Sigma, A5441).

Techniques: Activity Assay, Transfection, Negative Control, Luciferase, Control, Reverse Transcription, Over Expression, Knockdown, Staining, Positive Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity

doi: 10.1038/ncomms9093

Figure Lengend Snippet: ( a ) mRNA levels of ECAD (top), CAV1 (middle) and BCAT (bottom) in NRAS and NRAS-ΔPTEN mouse melanoma as measured by quantitative reverse transcriptase–PCR (fc, fold change). Experiments were performed three times. Error bars represent s.d. * P -value <0.05, ** P -value <0.001. Statistical significance was determined by Mann–Whitney test. ( b ) Western blot analysis of uncultured whole tumour lysates from NRAS and NRAS-ΔPTEN using antibodies against BCAT (total and pSer675), PTEN, CAV1, ECAD and β-actin. Western blot analyses were performed at least two times depending on antibody. ( c ) Immunofluorescence of serial NRAS and NRAS-ΔPTEN tumour sections, stained for ECAD, CAV1 and BCAT. Scale bar, 10 μm. xz projection from confocal image stacks of β-catenin-stained NRAS and NRAS-ΔPTEN tumours counterstained with 4,6-diamidino-2-phenylindole (DAPI). Arrowheads indicate nucleus. Scale bar, 16 μm. Immunofluorescence experiments for the NRAS and NRAS-ΔPTEN tumour cryosections were performed two times with similar results. ( d ) Interaction of CAV1 with β-catenin (BCAT) in NRAS and NRAS-ΔPTEN tumour samples. Cell lysates containing 400 μg of total proteins were subjected to IP with an anti-CAV1 antibody. The co-IP of β-catenin with CAV1 was detected by western blotting (WB) using anti-β-catenin and anti-CAV1 antibodies. Total protein input is shown. This experiment was performed twice. ( e ) mRNA levels of downstream β-catenin target genes c-MYC and CCND1 as measured from NRAS and NRAS-ΔPTEN mouse melanoma by quantitative reverse transcriptase–PCR (fc, fold change). Error bars represent s.d. ** P -value <0.01. Statistical significance was determined by Mann–Whitney test. ( f ) CAV1 and PTEN were detected by immunohistochemistry on a series of 50 human melanoma sections from the second cohort. Sections of patients were either AEC stained (red) for CAV1 (background image) or PTEN (foreground image, lower-right corner) and counterstained with haematoxylin (blue). Staining for CAV1 was scored as high or low, while the amount of PTEN as positive (pos) or negative (neg). Among 50 melanoma biopsies, 34 presented a low amount of CAV1 and a positive staining for PTEN (34/50). Scale bar, 100 μm.

Article Snippet: Membranes were probed with antibodies against ERK (Cell Signaling, 9102), pERK (Thr202/Thr204, Cell Signaling, 9106), CREB (Cell Signaling, 9192), pCREB (Cell Signaling, 9191S), PTEN (Cell Signaling, 9559), AKT (Cell Signaling, 2938), pAKT (Ser473, Cell Signaling 3787), GSK3-β (Santa-Cruz, sc-9166), pGSK3-β (Ser9, Cell Signaling, 9336), S6 (Cell Signaling, 2317), pS6 (Ser235/236) (Cell Signaling, 4857), β-catenin (Abcam, ab-6302), pβ-catenin (Ser675, Cell Signaling, 4176), pβ-catenin (Ser33-37/Thr41, Cell Signaling, 9561), pβ-catenin (Thr41/Ser45, Cell Signaling, 9565S), p16 (Santa-Cruz, sc-1661), CAV1 (Cell Signaling, 3238) and β-actin (Sigma, A5441).

Techniques: Reverse Transcription, MANN-WHITNEY, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity

doi: 10.1038/ncomms9093

Figure Lengend Snippet: ( a ) miR-199a-5p and miR-203 gene expression in NRAS and NRAS-ΔPTEN mouse tumour samples from miRNA expression arrays. ( b ) CAV1 mRNA expression post transfection with miRNA mimics for miR-199a and miR-203 in human Hs944T melanoma cells (fc, fold change). CAV1 knockdown after miRNA mimics overexpression from whole-cell protein lysates in duplicates was validated through western blotting (below). Statistical significance was determined by Mann–Whitney test. ( c ) miR-199a-5p and miR-203 gene expression in Hs944T after overexpression with miR mimic 199a-5p and 203, respectively (fold change). Error bars represent s.d. ** P -value <0.01. Statistical significance was determined by Mann–Whitney test. ( d ) miRNA levels as determined by quantitative reverse transcriptase–PCR (fold change) of mir-203 and mir-199a in Hs944T post siRNA-mediated knockdown of β-catenin (siBCAT) or conversely overexpression of BCAT. Statistical significance was determined by Mann–Whitney test. ( e ) CAV1 mRNA levels after either siBCAT or BCAT overexpression in Hs944T cells. Whole-cell protein lysates were analysed via western blot (see below). Error bars represent s.d. * P -value <0.05, ** P -value <0.01 and *** P -value <0.001. Statistical significance was determined by Mann–Whitney test. Experiments were performed in biological triplicates.

Article Snippet: Membranes were probed with antibodies against ERK (Cell Signaling, 9102), pERK (Thr202/Thr204, Cell Signaling, 9106), CREB (Cell Signaling, 9192), pCREB (Cell Signaling, 9191S), PTEN (Cell Signaling, 9559), AKT (Cell Signaling, 2938), pAKT (Ser473, Cell Signaling 3787), GSK3-β (Santa-Cruz, sc-9166), pGSK3-β (Ser9, Cell Signaling, 9336), S6 (Cell Signaling, 2317), pS6 (Ser235/236) (Cell Signaling, 4857), β-catenin (Abcam, ab-6302), pβ-catenin (Ser675, Cell Signaling, 4176), pβ-catenin (Ser33-37/Thr41, Cell Signaling, 9561), pβ-catenin (Thr41/Ser45, Cell Signaling, 9565S), p16 (Santa-Cruz, sc-1661), CAV1 (Cell Signaling, 3238) and β-actin (Sigma, A5441).

Techniques: Gene Expression, Expressing, Transfection, Knockdown, Over Expression, Western Blot, MANN-WHITNEY, Reverse Transcription

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Macrophage β-catenin is activated in HFD-induced MASH. Wild-type mice were provided with either a normal chow diet (NCD) or a high-fat diet (HFD) for 28 weeks. A Double immunofluorescence staining was performed on liver sections. Antibodies against F4/80 (displayed in green) and A-β-catenin (shown in red) were used. The cell nuclei were stained with DAPI (appearing blue). Five mice were included in each group. The scale bar represents 200 μm. B Liver macrophages were separated from mice that had been fed NCD or HFD. The mRNA level of Ctnnb1 (the gene encoding β-catenin) in liver macrophages of wild-type mice that had received NCD or HFD for 28 weeks was measured ( n = 5 samples/group). C Western blot analysis was carried out to detect the expression of A-β-catenin in liver macrophages from wild-type mice fed with NCD or HFD ( n = 5 samples/group). The data are presented as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Double Immunofluorescence Staining, Staining, Western Blot, Expressing, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Deficiency of myeloid β-catenin alleviates hepatic steatosis induced by HFD. A Serum ALT and AST levels were measured in β-catenin FL/FL and β-catenin M−KO mice following 28 weeks of normal chow diet (NCD) or high-fat diet (HFD) feeding ( n = 5 samples/group). B Liver-to-body weight ratios were examined in β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). C Liver triglyceride (TG) and total cholesterol (TC) levels were determined in β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). D Representative hematoxylin and eosin (H&E) staining of liver sections and the non-alcoholic fatty liver disease activity score (NAS) were obtained from β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). The scale bars represent 100 μm. E Representative Oil Red O-stained images of liver sections were taken from β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). The scale bar is 50 μm. The data are presented as mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Staining, Activity Assay, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Deficiency of myeloid β-catenin ameliorates MASH-associated liver fibrosis. A Representative Sirius Red staining of liver sections was shown from β-catenin FL/FL and β-catenin M−KO mice. These mice were fed with either a normal chow diet (NCD) or a high-fat diet (HFD) for 28 weeks ( n = 5 samples/group). Scale bars were 100 μm. B Immunofluorescence images of α-SMA + myofibroblasts in liver sections were obtained from NCD-fed or HFD-fed β-catenin FL/FL and β-catenin M−KO mice ( n = 5 samples/group). Scale bars measured 50 μm. C Relative mRNA levels of fibrogenic genes such as Acta2 , Col1α1 , Col3α 1, and Timp 1 were detected in the livers of β-catenin FL/FL and β-catenin M−KO mice that were fed with NCD or HFD ( n = 5 samples/group). Data were expressed as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Staining, Immunofluorescence, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Deficiency of myeloid β-catenin inhibits Ihh expression in macrophages in HFD-induced MASH. A Relative mRNA levels of Shh and Ihh in liver macrophages isolated from HFD-fed β-catenin FL/FL and β-catenin M−KO mice were determined. B Western blot was performed to analyze the expression of Ihh in liver macrophages from HFD-fed β-catenin FL/FL and β-catenin M−KO mice. C Double immunofluorescence staining was carried out on liver sections using antibodies against F4/80 (green) and Ihh (red). The nuclei were stained with DAPI (blue). There were five mice in each group. The scale bar represents 200 μm. The data are shown as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Expressing, Isolation, Western Blot, Double Immunofluorescence Staining, Staining, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Ihh signaling is crucial for macrophage β-catenin-mediated hepatic fibrosis in HFD-induced MASH. To restore Ihh expression in macrophages, mice were administered the Ihh plasmid via polyethylenimine nanoparticles, namely in vivo jetPEI-Man. A Representative Sirius Red staining images of liver sections from HFD-fed β-catenin FL/FL and β-catenin M−KO mice. These mice were either treated or not treated with jetPEI-Man-Ihh (jetPEI-Ihh) or jetPEI-Man-GFP (jetPEI-GFP) ( n = 5 mice per group). Scale bars are 100 μm. B Immunofluorescence images of α-SMA + myofibroblasts in liver sections from HFD-fed β-catenin FL/FL and β-catenin M−KO mice. These mice were either treated or not treated with jetPEI-Ihh or jetPEI-GFP ( n = 5 mice per group). Scale bars measure 50 μm. C Relative mRNA levels of fibrogenic genes such as Acta2 , Col1α1 , Col3α1 , and Timp1 in the livers of HFD-fed β-catenin FL/FL and β-catenin M−KO mice. These mice were either treated or not treated with jetPEI-Ihh or jetPEI-GFP ( n = 5 mice per group). Data are expressed as the mean ± standard deviation (SD). * P < 0.05, ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Expressing, Plasmid Preparation, In Vivo, Staining, Immunofluorescence, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Macrophage β-catenin induces Ihh expression and secretion. Bone marrow-derived macrophages (BMDMs) were treated with LPS (100 ng/ml) and PA (250 µM) or PBS for 12 h. A The mRNA level of Ctnnb1 in BMDMs following treatment with PA/LPS or PBS. B Western blot analysis was carried out to assess the expression of A-β-catenin in BMDMs after treatment with PA/LPS or PBS. C BMDMs underwent a ChIP-PCR assay using an anti-β-catenin or IgG antibody to detect the potential β-catenin binding site within the Ihh intron 1. D The mRNA level of Ihh in BMDMs isolated from β-catenin FL/FL and β-catenin M−KO mice. E The concentration of Ihh in the media of BMDMs isolated from β-catenin FL/FL and β-catenin M−KO mice was measured by ELISA. The data are presented as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Expressing, Derivative Assay, Western Blot, Binding Assay, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Macrophage β-catenin-induced Ihh promotes HSC activation. A The schematic illustration of the co-culture model that utilizes bone marrow-derived macrophages (BMDMs) and primary murine hepatic stellate cells (HSCs). B The relative mRNA levels of fibrogenic genes such as Acta2 , Col1α1 , and Col3α1 in HSCs were measured after co-culturing with BMDMs from β-catenin FL/FL and β-catenin M−KO mice. C Immunofluorescence pictures of α-SMA expression in HSCs were obtained after co-culturing with BMDMs from β-catenin FL/FL and β-catenin M−KO mice. The scale bars represent 100 μm. D Immunofluorescence images of α-SMA expression in HSCs were captured after co-culturing with BMDMs transfected with the Lv-β-catenin plasmid or the control vector (Lv-GFP). The scale bars are 100 μm. E Immunofluorescence images of α-SMA expression in HSCs were taken after co-culturing with Lv-β-catenin-transfected BMDMs, either in the presence or absence of Ihh neutralizing antibody. The scale bars measure 100 μm. F The mRNA levels of Gli1 , Gli2 , and Gli3 in HSCs were determined after co-culturing with BMDMs from β-catenin FL/FL and β-catenin M−KO mice. The data are presented as the mean ± standard deviation (SD). * P < 0.05, ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Activation Assay, Co-Culture Assay, Derivative Assay, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Schematic representation of the macrophage β-catenin-Ihh axis in modulating the activation of hepatic stellate cells and fibrosis in MASH. The consumption of a high-fat diet (HFD) led to the activation of β-catenin in liver macrophages. The activation of macrophage β-catenin triggered the expression and secretion of Ihh, which in turn facilitated the activation of hepatic stellate cells and fibrosis in MASH

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Activation Assay, Expressing

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: β-catenin is essential for CUG2-induced CSC-like phenotypes. (A) A549-VEC and A549-CUG2 cells were separated into cytosolic and nuclear fractions. β-catenin expression was determined by performing western blot analysis with an anti-β-catenin antibody. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. The cytosolic and nuclear fractions were confirmed by detecting actin and Sp1, respectively. (B) Total RNAs (3 µ g) were isolated from the A549-VECand A549-CUG2 cells, and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (C) A549-VEC and A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector and were harvested at 48 h after the transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results are an average of three experiments; bars indicate the means ± SD ( *** P<0.001, A549-VEC vs A549-CUG2 cells). (D) Transfection of efficiency of β-catenin siRNAs (#1 or #2) was confirmed by western blot analysis. (E) Migration of A549-CUG2 cells was measured with the wound healing assay at 24 h after control or β-catenin siRNAs (#1 and #2) transfection. Wound closure areas were monitored using a phase-contrast microscope at ×100 magnification, and the assay was repeated twice. (F) Invasion of A549-CUG2 cells transfected with control or β-catenin siRNAs (#1 and #2) was determined using the 48-well Boyden chamber. The chamber was assembled using Matrigel-coated polycarbonate filters. Scale bar indicates 100 µ m, and the assay was repeated twice. Each assay was performed in triplicate, and error bars indicate the means ± SD ( *** P<0.001, control vs. β-catenin siRNA). (G) A549-CUG2 cells (1,000 cells per well) transfected with control or β-catenin siRNAs (#1 and #2) were seeded in ultra-low attachment plates for 2, 4, or 6 days. The assay was performed in triplicate, and error bars indicate the means ± SD ( ** P<0.01 and *** P<0.001, control vs. β-catenin siRNA). Scale bars indicate 50 µ m. CUG2, cancer-upregulated gene 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Migration, Wound Healing Assay, Microscopy

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-induced β-catenin phosphorylation is unique and differs from that induced by Wnt3a. (A) A549-VEC and A549-CUG2 cells were treated with wortmannin (10 µ M), H89 (10 µ M), PP2 (2.5 µ M) and DMSO (mock) in the presence of Wnt3a (100 ng/ml) or PBS as a vehicle for 12 h, and cell lysates were prepared. Proteins present in the cell lysates were separated by performing SDS-PAGE on 10% gels, and β-catenin and α-actin were detected using corresponding antibodies. (B and C) Proteins present in the lysates of A549-VEC cells treated with Wnt3a (100 ng/ml) or PBS, and of A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were determined using antibodies against phosphorylated Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (D) Lysates of A549-VEC and A549-CUG2 cells treated with a control or GSK3β siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. GSK3β and phosphorylated GSK3β were detected by performing western blot with corresponding antibodies. CUG2, cancer-upregulated gene 2; GSK3β, glycogen synthase kinase 3β.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Control, Western Blot

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-activated NEK2 is responsible for the phosphorylation of β-catenin at Ser33/Ser37. (A) Lysates of A549-VEC and A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels, and NEK2 was detected by performing western blot analysis with anti-NEK2 antibody. (B) Levels of NEK2 and β-catenin phosphorylated at Ser33/Ser37 (p-β-catenin; p-Ser33/Ser37) in A549-VEC and A549-CUG2 cells were determined by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) β-catenin was pulled down from the lysates of A549-VEC and A549-CUG2 cells by using an anti-β-catenin antibody. NEK2 present in the immunoprecipitates was detected using an anti-NEK2 antibody, and β-catenin present in the immunoprecipitates was detected as a loading control by using an anti-β-catenin antibody. (D) NEK2 was pulled down from A549-VEC and A549-CUG2 cells by using an anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay, including immunoprecipitates as NEK2 kinase, recombinant GST-β-catenin as a substrate, ATP, and a reaction buffer, was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by determining the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing immunoblotting, and β-catenin levels were examined as a loading control. (E) Lysates of A549-CUG2 cells transfected with the control or NEK2 siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (F) Following transfection with control or NEK2 siRNA#1, the levels of p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells were detected by performing immunofluorescence microscopy with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Recombinant, Incubation, Activity Assay, Transfection

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment destabilizes β-catenin in a proteasome- and PKCα- dependent manner. (A) A549-CUG2 cells were treated with CGK062 (0, 1, 10, 50 and 100 µ M) for 24 h, and β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (B) A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector in the presence of CGK062 (50 µ M) and were harvested at 48 h following transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results shown are an average of 3 experiments; bars indicate the means ± SD ( *** P<0.001, 0 vs. 10 µ M, 0 vs. 50 µ M). (C) Total RNAs (3 µ g) were isolated from A549-CUG2 cells treated with or without CGK062 (50 µ M), and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (D) A549-CUG2 cells were treated with MG132 (1 µ M) for 8 h in the absence or presence of CGK062 (50 µ M) and were harvested. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (E) A549-CUG2 cells were treated with BMI (7.5 µ M), a specific PKCα inhibitor, or were transfected with a control and PKCα siRNAs (#1 or #2) in the absence or presence of CGK062 (50 µ M) for 24 h. Transfection efficiency of PKCα siRNAs (#1 or #2) was confirmed by western blot analysis. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. CUG2, cancer-upregulated gene 2; PKC, protein kinase C.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment decreases NEK2 expression and kinase activity in A549-CUG2 cells. (A) Lysates of A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 µ M) for 24 h were separated by performing SDS-PAGE on 10% gels. The expression of NEK2, β-catenin, E-cadherin, N-cadherin and vimentin was detected by performing western blot analysis with corresponding antibodies. (B) Levels of NEK2 and p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO as Mock were detected by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) NEK2 was pulled down from A549-VEC and A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO by using the anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by detecting the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing western blot analysis, and β-catenin levels were examined as a loading control. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Activity Assay, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Incubation, Phospho-proteomics